These studies are intended to establish Tetrahymena as an alternative expression system for use in high-throughput structural studies, and for large-scale production of membrane proteins with importance in human health. Towards that end, we will attempt to express 3 model membrane proteins in this system using a unique and versatile high-copy number cloning vector. The vector (now under construction) takes advantage of a novel biological feature of Tetrahymena in which the ribosomal genes are amplified to ~ 18,000 copy number as part of an autonomously replicating palindromic minichromosome. Using a similar vector that lacks a multicloning site and affinity tags, we have been able to express a heterologous GPI-anchored membrane protein in Tetrahymena to levels greater than 3% of total cell protein. We now want to validate this approach for important transmembrane proteins in humans. This includes the human epidermal growth factor receptor (a G-protein coupled receptor) and the ionotropic glutamate receptor GluRG (which acts as a channel protein). Tetrahymena has a number of distinct features which set it apart from more conventional protein expression systems and which make it ideal as a vehicle for the production of recombinant eucaryotic membrane proteins. A successful outcome, in this case overexpression of functional proteins, will provide proof-of-principal for the use of this system and make it an attractive tool for use by the biopharmaceutical industry. Our Small Business Partner, Tetragenetics Inc, seeks to commercialize on this technology and make it available to researchers in both the academic and industry arenas through protein expression kits and contract expression services. Eucaryotic membrane proteins contribute to an array of vital cell functions and are essential to human health. The technology described here is intended to better understand how these proteins function, leading ultimately to strategies for the treatment of serious disorders ranging from cancer to stoke. [unreadable] [unreadable]